This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Nitrogenase is believed to utilize two unique metalloclusters in the reduction of atmospheric nitrogen: FeMoco and the P-cluster. The assembly of these metalloclusters in vivo is a complex bioinorganic process of significant current interest. The genetic basis of FeMoco assembly has been largely established, but an understanding of P-cluster formation is in its nascency. In either case, little is known about the molecular mechanism by which these clusters are constructed. We propose to directly probe the details of FeMoco and P-cluster assembly by using Fe K-edge XAS to determine the structures and electronic states of the precursors to these clusters on proteins identified as precursor carriers. In an initial study of P-cluster formation, we analyzed FeMoco-deficient MoFe proteins resulting from nifH and nifB deletion strains and were able to confirm the presence of a wild-type [Fe8S7] P-cluster structure in the delta nifB MoFe protein and identify a novel [Fe-S] cluster in the delta nifH MoFe protein (M.C. Corbett, et al. J. Biol. Chem. 279, 28276 (2004)). Through this study, we developed a model that suggests the P-cluster is formed by the condensation of two [Fe4S4] fragments, possibly concomitant with a Fe protein (NifH) induced conformational change. We propose to test our theory regarding conformational change through a comparative SAXS study of the delta nifH MoFe protein with wild-type and delta nifB MoFe proteins. Further, we propose to enhance our model of P-cluster biosynthesis through the study of delta nifH MoFe protein incubated in the presence of other proteins suspected to effect P-cluster formation in vivo. In a related set of experiments, we plan to examine the mechanism of FeMoco biosynthesis through the study of NifEN, a MoFe protein homologue believed to be a site of FeMoco assembly, alone and after incubation with the components required for complete FeMoco formation.